siRNA Delivery via Cross‐Linked Gelatin Microparticles Enables Targeted Modulation of Osteogenic‐Vascular Cross‐Talk: An Advanced Human 3D in Vitro Test System for Therapeutic siRNA

Small interfering RNAs (siRNAs) have drawn particular attention for their ability to transiently and sequence-specifically silence target genes, not only for systemic but also for localized application. For bone regeneration, targeting inhibitory regulators by siRNAs offers opportunities to improve osteogenic-angiogenic coupling. Conventional experimental models often oversimplify this interaction as they fail to capture these multicellular tissue dynamics. To address this, we established a human three-dimensional co-culture model composed of osteogenic and vascular microtissues embedded in fibrin hydrogels to investigate siRNA effects on microtissue interaction. Local siRNA delivery to microtissues was achieved by oligomer-stabilized calcium phosphate nanoparticles (CaP-NP) loaded onto cross-linked gelatin microparticles (cGM). siRNA/CaP-NP-loaded cGM were assembled with human mesenchymal stem cells (hMSCs) to microtissues. This approach was demonstrated by silencing two antagonists with distinct expression profiles: Chordin, a low-abundance BMP inhibitor, and WWP-1, a highly expressed E3 ligase. Only Chordin siRNA improved the osteogenic-vascular cross-talk, whereas WWP-1 siRNA effects were limited to osteogenic effects. Next-generation sequencing (NGS) supported these results. We demonstrate that this co-culture platform permits systematic investigation of siRNA-mediated modulation of osteogenic-endothelial interactions, offering a relevant human model for preselecting therapeutic siRNA targets to advance vascularized bone tissue regeneration.